JP2008228640A - Method for concentrating and/or refining sugar chain compound - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for economically and industrially concentrating and/or refining a sugar chain compound obtained by a sugar chain primer extension technique or the like. <P>SOLUTION: The method includes the following process: animal cells are cultured under such conditions that a desired sugar chain compound is obtained in a cell culture supernatant by the sugar chain primer extension technique, and a solid adsorbent is brought into contact with the resultant culture supernatant, wherein the solid adsorbent is a styrenic or methacrylic synthetic adsorbent. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a method for concentrating and / or purifying a sugar chain compound, and more specifically, concentrating and / or purifying a sugar chain compound obtained by a method of extending a sugar chain primer using cells from a cell culture or culture medium supernatant. Regarding the method.

  Sugar chains are complex in structure, and development for industrial use is delayed compared to DNA and proteins, which are the same biomolecules. However, in recent years, research on sugar chains has progressed rapidly, and the search for industrial use has been promoted. In particular, new facts about the involvement of sugar chains in cell differentiation, canceration, immune reaction, etc. are being revealed. For example, sugar chains on the cell surface are involved in important processes such as cell differentiation, canceration, and immune reaction in vivo by interacting with proteins and lipids. In addition, it has become clear that sugar chains play an important role in cell recognition, adhesion and signal transduction in the cell surface layer.

  Examples of methods for synthesizing sugar chains include chemical synthesis methods, decomposing enzyme methods, and glycosyltransferase methods. However, each process is complicated, yield is low, and raw materials used are limited. There are disadvantages such as. Under such circumstances, recently, sugar chain synthesis technology utilizing the function of animal cells has attracted attention.

  In glycan synthesis using the function of animal cells, products that have undergone glycan elongation when added to the medium, such as by adding a glycan primer, which is a precursor of the glycan synthesis pathway, to the medium The sugar chain compound is secreted extracellularly. Here, as the sugar chain primer, a compound having a structure in which an alkyl chain is bonded to an oligosaccharide is basically used, but a compound having a structure in which various functional groups such as an azide group are added to the terminal is used. You can also. However, since such sugar chain primers are usually toxic to cells, the concentration of addition must be limited, and the concentration of the resulting sugar chain compound does not necessarily become high. Therefore, when utilizing the obtained sugar chain compound, it is necessary to concentrate together with separation and purification from the culture medium.

  However, there has been no method for concentrating and / or separating and purifying a large amount of sugar chain compounds required for sugar chain synthesis utilizing the function of animal cells so far. Concentration and separation methods using HPLC or HPTLC have been performed (for example, Patent Document 1, Non-Patent Document 1, Non-Patent Document 2, Non-Patent Document 3, and Non-Patent Document 4).

JP 2000-247992 A M. C. Z. Kasuya et al., Carbohydrate Research 329 (2000) 755-763 Johannes Muthing, Methods in Enzymology, Vol312, Part B, 45-64 Stephan Ladisch and Ruixiang Li, Methods in Enzymology, Vol312, Part B, 135-145 Robert K. Yu and Toshio Ariga, Methods in Enzymology, Vol312, Part B, 115-134

  The C18-based solid phase cartridge currently used for concentration and / or purification of sugar chain compounds or the method using HPLC or HPTLC is not suitable for processing a large amount of sample because of the high cost of the cartridge or the apparatus. Thus, there is no method for concentrating and / or purifying a large amount of sugar chain compounds efficiently and inexpensively, and development of such a method is strongly desired.

  Accordingly, an object of the present invention is to provide a method for concentrating and / or purifying a sugar chain compound obtained by a sugar chain primer extension method or the like from cells in a large amount at low cost from a cell culture or its supernatant. .

  As a result of intensive studies, the present inventor has found that the above object can be achieved by the following method for concentrating and / or purifying sugar chain compounds produced by cells, and has completed the present invention.

  That is, the present invention is a method for concentrating and / or purifying a sugar chain compound from a cell culture, which comprises the step of bringing such a cell culture or a cell culture supernatant into contact with a solid adsorbent. I will provide a.

  The method may further comprise the step of eluting the sugar chain compound from the solid adsorbent that has been contacted with the cell culture or the cell culture supernatant.

  The elution step is performed using at least one eluent selected from the group consisting of methanol, ethanol, isopropanol, butanol, acetone, and acetonitrile.

  The solid adsorbent may be at least one selected from the group consisting of a styrene synthetic adsorbent and a methacrylic synthetic adsorbent.

  The cell culture can be obtained by adding a sugar chain primer to cells and culturing the cells.

  The cell can be an animal cell.

    The present invention also provides a method for separately concentrating and / or purifying a plurality of types of sugar chain compounds from a cell culture, comprising contacting the cell culture or the cell culture supernatant with a solid adsorbent; and And elution of a plurality of sugar chain compounds with different concentrations of an eluent from a cell culture or a solid adsorbent in contact with the cell culture supernatant. In this method, a medium-derived component, a neutral sugar chain compound, and an acidic sugar chain compound can be particularly preferably separated, purified and / or concentrated.

  The eluting step is performed using at least one eluent selected from the group consisting of methanol, ethanol, isopropanol, butanol, acetone, and acetonitrile, and the solid adsorbent is a styrene synthetic adsorbent and a methacrylic adsorbent. It may be at least one selected from the group consisting of synthetic adsorbents.

  As described above, by applying the present invention, it is possible to economically produce a sugar chain compound produced by extending from a sugar chain primer using a cell, particularly an animal cell, from a cell culture or its supernatant. And it can be concentrated and / or purified industrially easily. Therefore, it becomes possible to prepare a large amount of useful animal cell-derived sugar chain compounds including human-type sugar chains at a low cost, and it is possible to rapidly put into practical use the development of products using sugar chains.

  Embodiments of the present invention will be described below.

  The cell culture of the present invention is preferably obtained by culturing animal cells under conditions such that a sugar chain compound can be obtained. Animal cells are not particularly limited, and various animal cells can be used depending on the type of the desired sugar chain compound. Examples of animal cells include mammalian cells such as human origin, mouse origin, monkey origin, rat origin and the like, in particular B16 mouse melanoma cells, rat PC12 cells, mouse Neuro2a cells, rat RBL-2H3 cells, human MOLT- 4 cells, human HL-60 cells, monkey kidney-derived Vero cells and the like can be suitably used. Culturing is performed according to normal animal cell culture conditions.

  The conditions under which the sugar chain compound can be obtained include, for example, that the above animal cells are previously cultured in a culture medium such as DMEM / F12 at a temperature between room temperature and 45 ° C. for a period of 1 hour to 70 hours. After culturing, the medium is replaced with a medium supplemented with a saccharide primer, and further culturing is performed to carry out a glycan elongation reaction.

Here, the sugar chain primer can be arbitrarily selected depending on the cell type used and the desired sugar chain structure. Although not limited, Examples of preferable sugar chain primers include compounds represented by the formula (I): (G1) x (G2) y (G3) z-L-X. Here, in the formula, G 1, G 2 and G 3 are each independently a monosaccharide residue having a cyclic structure or a derivative thereof, and L is —O— (CH ₂ ) n —, —S— (CH ₂ ). n-, and -NH- (CH ₂₎ a linking group selected from n-, X is, -N _3, -NH _2, is selected -OH, -COOH, and from -C ₁ -C ₄ alkyl X, y and z are each independently an integer of 0 to 10, preferably 0 to 5, more preferably 0 to 3, and all of x, y and z are simultaneously 0. Never. N is an integer of 8 to 20, preferably 8 to 16, and particularly preferably 12. The monosaccharide is not limited, but pentose and hexose are preferably used. Either aldose or ketose can be used.

  When such a sugar chain primer is added to the culture medium of various cells and the cells are cultured, the cells take up the primer, add sugar to the sugar chain part to glycosylate, and introduce the sugar addition product into the cell. Secretes extracellularly without accumulating. The added sugar varies depending on the cell.

  The resulting cell culture or the supernatant obtained by removing cells from the cell culture contains a sugar chain compound that is a product obtained by extending from a sugar chain primer. The sugar chain compound can be concentrated and / or purified by bringing the solid adsorbent into contact with the cell culture or the cell culture supernatant.

  The contact is performed, for example, by adding a solid adsorbent directly to the cell culture or the cell culture supernatant and shaking for 30 minutes or longer. Alternatively, a solid adsorbent is packed in advance in a column or the like, and the cell culture or the supernatant of the cell culture is passed through the packed column.

  In the present invention, the solid adsorbent after completion of the contacting step is contacted with an eluent having an appropriate concentration to desorb the sugar chain compound from the solid adsorbent, and the desorbed sugar chain compound is put into the eluent. An elution step may be performed to elute. The eluent is not limited, but may be at least one selected from the group consisting of methanol, ethanol, isopropanol, butanol, acetone, and acetonitrile. In this process, the sugar chain compound is desorbed from the adsorbent by bringing an eluent such as an organic solvent into contact with the adsorbent adsorbed by the hydrophobic interaction between the sugar chain compound and the adsorbent. In this step, the compound is eluted in an organic solvent.

  Here, the solid adsorbent may be at least one selected from the group consisting of a styrene synthetic adsorbent and a methacrylic synthetic adsorbent. In addition, a phenol-based synthetic adsorbent can also be used. Examples of the styrene synthetic adsorbent that adsorbs the sugar chain compound in the culture or the culture supernatant include those having a styrene-divinylbenzene copolymer as a resin matrix. Specific examples of the styrene-based synthetic adsorbent include Diaion (registered trademark, hereinafter the same) HP20, HP21, Sepabeads (registered trademark, the same) SP207, SP825, SP850, SP875, manufactured by Rohm and Haas, manufactured by Mitsubishi Chemical Corporation. Amberlite (registered trademark, the same shall apply hereinafter) XAD4, XAD16, XAD1180, XAD2000, Bayer VPOC1062, VPOC1064, and the like. Further, as a methacrylic synthetic adsorbent, Diaion (registered trademark, the same shall apply hereinafter) HP1MG, HP2MG, etc. manufactured by Mitsubishi Chemical Corporation can be used. According to the study by the present inventors, it has been found that the styrene-based and methacrylic synthetic adsorbents particularly favorably adsorb sugar chain compounds.

  The present invention also provides a method for separately concentrating and / or purifying a plurality of types of sugar chain compounds from a cell culture, comprising contacting the cell culture or the cell culture supernatant with a solid adsorbent; and A method comprising a step of eluting a plurality of sugar chain compounds with different concentrations of an eluent from a cell culture or a solid adsorbent contacted with a cell culture supernatant. The derived component, neutral sugar chain compound, and acidic sugar chain compound can be particularly preferably separated, purified and / or concentrated.

  That is, the sugar chain compound can be separated and purified by the difference in the adsorptive power of the synthetic adsorbent. For example, a substance having a large hydrophobic interaction can efficiently adsorb an acidic sugar chain compound having a high polarity, while a neutral sugar chain compound having a low polarity is difficult to elute. On the other hand, those having a small hydrophobic interaction are easily eluted with a highly polar acidic sugar chain compound with a highly polar solvent, whereas a neutral sugar chain compound with low polarity is also easily eluted. The sugar chain compound can be separated and purified by utilizing the adsorptive power of the synthetic adsorbent as described above.

  For example, when a sugar chain compound is adsorbed using Diaion HP20 as an adsorbent, medium-derived components can be eluted with 50% concentration of methanol, and acidic sugars such as GM3 can be eluted with 70% concentration of methanol. A chain compound can be eluted, and neutral sugar chain compounds such as 12-azidododecyl-β-lactoside, which is a neutral sugar chain primer, and Gb3 and Gb4 can be eluted with 100% concentration of methanol.

  That is, two or more types of eluents having different polarities can be used, and these eluents can be brought into contact with the synthetic adsorbent in descending order of polarity. In this way, by gradually bringing the eluent into contact with the one having a high polarity to the one having a low polarity, the elution solution can be eluted in order from the high polarity compound, and can be separated according to the polarity of the sugar chain compound.

  In addition, when Diaion SP207, which has a strong hydrophobic interaction or strong adsorptive power, is used as the adsorbent, the neutral sugar chain primer 12-azidododecyl-β-lactoside is completely intact even when 100% methanol is used. It can be used to remove neutral sugar chains such as sugar chain primers.

  The neutral sugar chain primer, 12-azidododecyl-β-lactoside, can be removed by adding the same amount of ethyl acetate to the culture supernatant and shaking to extract the ethyl acetate layer. . Therefore, removing 12-azidododecyl-β-lactoside, which is a neutral glycan primer by extraction with ethyl acetate, before bringing the culture supernatant into contact with the adsorbent reduces the load in the adsorption and elution steps. It can be reduced.

  A sugar chain primer can be prepared and used by combining lactose, N-acetylglucosamine, etc. with a dodecyl group. Furthermore, if a functional group such as an azide group is introduced into the aglycon moiety, the moiety and another compound can be bound and immobilized by a covalent bond or the like in a later operation.

  Specific examples of the method for purifying and concentrating the sugar chain compound of the present invention are shown below in the embodiments, but the present invention is not limited thereto.

First, after performing glycosylation using a Lewis acid boron trifluoride etherate (BF ₃ · OEt ₂ ) and an azide-introduced alcohol as a catalyst, a deacetylation reaction is carried out, so that a glycan primer: 12- Azidododecyl-β-lactoside was obtained (Murozuka, Y., Kasuya, MC, Kobayashi, M., Watanabe, Y., Sato, T. and Hatanaka, K. 2005, Chemistry and Biodiversity, 2, 1063, Kasuya, MC , Wang, L., Lee, YC, Mitsuki, M., Nakajima, N., Sato, T., Hatanaka, K., Yamagata, S. and Yamagata, T. 2000, Carbohydr. Res., 329, 755. )

To 100mm dish were plated in suspension Vero cells derived from kidney of mouse melanoma B16 cells and monkey cells number 2 × ¹⁰ 6 cells into DMEM / F12 medium (Invitrogen) containing 10% FBS 7ml each, CO ₂ Pre-culture was performed for 48 hours at 37 ° C. in an incubator.

After preculture, the medium was replaced with DMEM / F12 medium supplemented with 12-azidododecyl-β-lactoside and 1% ITSX (GIBCO) to a final concentration of 50 μM, and further 37 ° C. in a CO ₂ incubator. Incubated for 48 hours to carry out a sugar chain elongation reaction. After the sugar chain elongation reaction, the medium was collected, and the dish surface was further washed with 1.5 ml of PBS to collect the whole medium supernatant.

  The obtained culture supernatant was used for concentration and purification in the present invention. First, after performing ethyl acetate extraction treatment, Diaion HP20 was used as the styrene synthetic adsorbent, and 50% methanol, 70% methanol, and 100% methanol were used as the eluent in this order.

Specifically, the sugar chain compound was concentrated and purified as follows.
(1) The saccharide primer removal operation by extraction with the same amount (8.5 ml) of ethyl acetate was performed twice on the medium supernatant (7 ml) + the amount of washing solution (1.5 ml).
(2) The solution from which the sugar chain primer was removed with ethyl acetate was passed through a column filled with a 5 ml styrene synthetic adsorbent (Diaion HP20) (adsorbent contact step).
(3) After completion of the flow, the styrene synthetic adsorbent was washed with 4 column volumes of water. Next, 20 ml each of 50% methanol, 70% methanol, and 100% methanol was sequentially passed as an eluent (elution process).

  Each eluent was concentrated under reduced pressure, dried, and analyzed by HPTLC. The results are shown in FIG. As a developing solvent, chloroform / methanol / 0.25% KCl aqueous solution (5: 4: 1) was used, and color development was performed with resorcinol.

  The band slightly below the center of FIG. 1 (A) is a band derived from an acidic sugar that is a sugar compound produced using B16 cells. The upper band is a band derived from a sugar chain primer. As is clear from FIG. 1 (A), the concentration and / or purification of acidic sugar by the styrenic synthetic adsorbent can be achieved by the conventional Sep-PakC using any of the 70% methanol and 100% methanol eluents. It was proved that it can be equal to or higher than when elution is performed under the same conditions using -18 columns (Waters). The band around the center in FIG. 1B is a band derived from a neutral sugar, which is a sugar compound generated using Vero cells. As is clear from FIG. 1B, when a 100% methanol eluent is used, neutral sugar concentration and / or purification by a styrenic synthetic adsorbent is performed using a conventional Sep-PakC-18 column ( It was proved that when water was used, it was possible to achieve the same or better than when elution was performed under the same conditions.

150 100 mm dishes were each seeded with 2 × 10 ⁶ cells of mouse melanoma B16 cells suspended in 7 ml of DMEM / F12 medium containing 10% FBS at 37 ° C. in a CO ₂ incubator. Pre-culture was performed for 48 hours. After preculture, the medium was replaced with DMEM / F12 medium supplemented with 12-azidododecyl-β-lactoside and 1% ITSX (GIBCO) to a final concentration of 50 μM, and further 37 ° C. in a CO ₂ incubator. Incubated for 48 hours to carry out a sugar chain elongation reaction. After the sugar chain elongation reaction, the medium was collected, and the dish surface was washed with PBS to collect the whole medium supernatant.

The obtained culture supernatant was used for concentration and purification in the present invention. In this case, 50 g of styrene synthetic adsorbent Diaion HP20 was first added to the culture supernatant and shaken for 16 hours to adsorb the sugar chain compound to the adsorbent (adsorbent contact step).
Next, after washing with 100 ml of water five times, 50% concentration methanol, 70% concentration methanol, and 100% concentration methanol were sequentially passed through each 100 ml four times.

  The eluate with 50% methanol and 70% methanol is concentrated by a rotary evaporator and then re-adsorbed on the adsorbent in a column filled with 50 ml styrene synthetic adsorbent (Diaion HP20). After passing an appropriate amount of 40-50% methanol, GM3 type sugar chain compound as the target sugar chain compound was eluted with 200 ml of 70% methanol.

  On the other hand, the eluate with 100% methanol was concentrated by a rotary evaporator and then dried. After 3 ml of DMSO was added to the dried product and dissolved, 100 ml of water was added, followed by re-adsorption to the adsorbent in a column filled with 50 ml of a styrene synthetic adsorbent (Separbeads SP207). After washing with 200 ml of water, the GM3-type sugar chain compound as the target sugar chain compound was eluted with 200 ml of 70% strength methanol.

  The eluate of each target sugar chain compound GM3 is concentrated under reduced pressure, dried, and analyzed by HPTLC. FIG. As a developing solvent, chloroform / methanol / 0.25% KCl aqueous solution (5: 4: 1) was used, and color development was performed with resorcinol. After all the eluate was concentrated under reduced pressure and dried, GM3 type sugar chain compound was obtained as 18.5 mg of white powder.

  In order to examine the ability of various types of styrene synthetic adsorbents and methacrylic synthetic adsorbents to concentrate and / or purify sugar chain compounds, the following experiments were also conducted.

  BALL-1 cells were precultured in the same manner as in Example 1 or 2, and then replaced with a medium supplemented with 12-azidododecyl-β-lactoside. The cells were further cultured and subjected to a sugar chain elongation reaction. After the sugar chain elongation reaction, the medium was collected.

  Concentration and purification in the present invention were performed using the obtained culture medium (four of the same). First, each of the washed adsorbents (styrene HP20, SP850, SP207, methacrylic HP2MG) was passed through a column packed with 5 ml each. Subsequently, after washing with 10 ml of water, elution was performed using methanol of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100% concentration as an eluent.

  Each eluent was concentrated under reduced pressure, dried, and analyzed by HPTLC. The results are shown in FIG. As a developing solvent, chloroform / methanol / 0.25% KCl aqueous solution (5: 4: 1) was used, and color development was performed with resorcinol.

  In all of FIGS. 3 (A) to (E), the band slightly below the center is a band derived from acidic sugar, which is a sugar compound produced using BALL-1 cells. The upper band is a band derived from a sugar chain primer. As is clear from this figure, the concentration varies depending on the type of adsorbent used, but when methanol is used as the eluent, concentration and / or purification of acidic sugar can be achieved with either styrene synthetic adsorbent or methacrylic synthetic adsorbent. It has been proved that when a conventional Sep-PakC-18 column (Waters) is used, it can be equivalent to or more than when elution is performed under the same conditions. (S indicates the sample before fractionation)

(Comparative Example 1)
10 ml of BALL-1 cell culture obtained by the same method as in Example 1 was applied to a column (inner diameter 1.5 cm, length 15 cm) packed with 20 ml of DEAE-Sephadex A-25 (acetic acid type). Then flush 5 column volumes of CHCl ₃ / MeOH / H ₂ O (5: 10: 1) and then elute with 5 column volumes of CHCl ₃ / MeOH / 1M sodium acetate (5: 10: 1) Each fraction was desalted with Sep-Pak and subjected to HPTLC analysis. The result is shown in FIG. As is clear from FIG. 4, the band of the sugar chain compound obtained before separating the non-adsorbed fraction and the adsorbed fraction by DEAE could only be recovered by DEAE-Sephadex at a low recovery rate. .

It is the result of having analyzed the thing which concentrated and isolate | separated the sugar_chain | carbohydrate compound produced | generated by B16 cell and Vero cell by HP20 by HPTLC. It is the result of having analyzed the thing which refine | purified the sugar_chain | carbohydrate compound produced | generated by B16 cell by HP20 by HPTLC. It is the result of analyzing what refine | purified the sugar_chain | carbohydrate compound produced | generated by the BALL-1 cell with various synthetic adsorption agents by HPTLC. It is the result of analyzing what refine | purified the sugar_chain | carbohydrate compound produced | generated by the BALL-1 cell with the ion exchange resin by HPTLC.

Claims (9)

A method for concentrating and / or purifying a sugar chain compound from a cell culture comprising:
A method comprising the step of contacting the cell culture or a supernatant of the cell culture with a solid adsorbent. The method for concentrating and / or purifying a sugar chain compound according to claim 1, further comprising a step of eluting the sugar chain compound from the solid adsorbent contacted with the cell culture or the cell culture supernatant. The sugar chain compound according to claim 2, wherein the eluting step is performed using at least one eluent selected from the group consisting of methanol, ethanol, isopropanol, butanol, acetone, and acetonitrile. How to purify. The sugar chain compound according to any one of claims 1 to 3, wherein the solid adsorbent is at least one selected from the group consisting of a styrene synthetic adsorbent and a methacrylic synthetic adsorbent. Method. The method for concentrating and / or purifying a sugar chain compound according to any one of claims 1 to 4, wherein the cell culture is obtained by adding sugar chain primers to cells and culturing. The method for concentrating and / or purifying a sugar chain compound according to any one of claims 1 to 5, wherein the cell is an animal cell. The method for concentrating and / or purifying the sugar chain compound according to any one of claims 1 to 6, further comprising a step of removing the sugar chain primer with ethyl acetate. A method for concentrating and / or purifying a plurality of types of sugar chain compounds separately from a cell culture,
Contacting the cell culture or cell culture supernatant with a solid adsorbent; and different the plurality of sugar chain compounds from the solid adsorbent contacted with the cell culture or cell culture supernatant. Elution with a concentration of eluent, respectively. The eluting step is performed using at least one eluent selected from the group consisting of methanol, ethanol, isopropanol, butanol, acetone, and acetonitrile, and the solid adsorbent is a styrene synthetic adsorbent and a methacrylic adsorbent. The method according to claim 8, wherein the method is at least one selected from the group consisting of synthetic adsorbents.

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